Biomolecule Immobilisation Using Atmospheric Plasma Technology

ABSTRACT

The present invention is related to method for immobilising a biomolecule on a surface by generating and maintaining an atmospheric pressure plasma, said method comprising the steps of: introducing a sample in the space between two electrodes, a mixed atmosphere being present between said electrodes, applying an alternating voltage to said electrodes for generating and maintaining a plasma in the volumetric space between the electrodes, said voltage having a profile as a function of time, defined by a sequence of time periods during which a positive or zero voltage is applied, alternated with time periods during which a negative or zero voltage is applied, and depositing a coating on a surface of said sample, characterised in that said mixed atmosphere comprises an aerosol comprising a reactive precursor and an aerosol comprising a biomolecule, both of which are deposited and immobilised during the depositing step.

FIELD OF THE INVENTION

The present invention is related to plasma techniques, involving theinclusion of biological molecules into a plasma deposited layer.

STATE OF THE ART

It is known in the art to apply functional groups to a surface viaplasma technology. In a second step, it is then possible to attachbiomolecules to said functional groups. The functional groups can beobtained by activation of polymers or by application of a cover layerwith functional groups. In most cases, the known technology relates toat least a two-step process.

DE19835869 describes the stabilisation of immobilised enzyme on asubstrate, especially a biosensor or bioreactor. The document mentionssimultaneous application of enzymes on a surface and application of apolymer layer. The technology used is gas-phase deposition, whichcreates a harsh environment for the biomolecules and leads to unwanteddegradation thereof.

EP0351950 relates to the use of plasma to immobilise protein onpolymeric surfaces, wherein a two-step process is used whereinbiomolecules are exposed to a low-pressure (vacuum) plasma. Applicationof biomolecules is done separately from application of polymerprecursors.

The described process is thus only applicable to polymer substrates.

EP1231470 describes a method for immobilising substances with plasmatechnology. Biomolecules are brought in contact with plasma in at leasta two-step process: an optional plasma polymer layer is applied to asurface followed by spreading the biomolecules on said surface andapplication in vacuo of a plasma polymer film on said biomolecules. Itis doubtful that the biomolecules retain their activities with thismethod, as they are covered by a thick polymer film.

WO 03/086031 describes an atmospheric plasma process comprising sprayingliquid precursors in a plasma causing polymerisation. No specificmention is made of biomolecules.

AIMS OF THE INVENTION

The present invention aims to provide a method to immobilisebiomolecules on a surface so as to be able to use said biomolecules inspecific interaction with other molecules of interest. The object of thepresent invention is thus to develop an entirely new, one-step processfor the immobilisation of proteins/enzymes or other biomolecules, whichis applicable on a large scale to surfaces of any kind. The newmethodology should offer several advantages over the classicalimmobilisation techniques, including a better reproducibility, highflexibility, broad applicability, straightforward processing and thushigh throughput rates. The new way of processing may in turn lead toentirely new applications that are not feasible with the currentstate-of-the-art technology.

SUMMARY OF THE INVENTION

The present invention is related to a method for immobilising abiomolecule on a sample surface by generating and maintaining a coldatmospheric pressure plasma, said method comprising the steps of:

-   -   introducing a sample in the space between a first and a second        electrode, a mixed atmosphere being present between said        electrodes,    -   applying an alternating voltage to said first and second        electrode for generating and maintaining a plasma in the        volumetric space between said electrodes, said voltage        alternating between a positive voltage for said first electrode        and a zero voltage for said second electrode, and a zero voltage        for said first electrode and a negative voltage for said second        electrode, and    -   depositing a coating on a surface of said sample,        wherein a reactive precursor and a biomolecule are deposited and        immobilised during the depositing step.

Preferably, the reactive precursor is a gas or a liquid in the form ofan aerosol.

Preferably, the biomolecule is selected from the group consisting of aprotein, a polynucleotide, a sugar, a lipid, a growth factor, a hormoneand a physiologically active substance.

The reactive precursor can be selected from the group consisting of ahydrocarbon, a fluorinated hydrocarbon and an organometallic compound ora combination thereof.

The mixed atmosphere can comprise helium, argon, nitrogen, air, carbondioxide, ammonium or a combination thereof.

The sample can comprise metal, ceramic or plastic materials, woven ornon-woven fibres, natural fibres or synthetic fibres or powders.

If necessary, the electrodes can be cooled to temperatures between 0° C.and 100° C.

In a first embodiment of the present invention, the mixed atmospherecomprises the reactive precursor and an aerosol comprising thebiomolecule.

In an alternative embodiment of the present invention, said methodfurther comprises the steps of:

-   -   applying a solution containing said biomolecule onto a sample        surface,    -   introducing said sample in the space between the first and        second electrode or in the afterglow of the plasma which is        maintained between the two electrodes, a mixed atmosphere being        present between said electrodes,    -   applying an alternating voltage to said first and second        electrode for generating and maintaining a plasma in the        volumetric space between said electrodes, said voltage        alternating between a positive voltage for said first electrode        and a zero voltage for said second electrode, and a zero voltage        for said first electrode and a negative voltage for said second        electrode, and    -   depositing a coating on a surface of said sample,    -   wherein said mixed atmosphere or its afterglow comprises the        reactive precursor, which is deposited onto the sample surface        during the depositing step.

The step of applying the solution containing the biomolecule onto asample surface is preferably selected from the group consisting ofspreading out of the solution followed by drying, adsorption andcovalent linking with or without making use of spacer molecules.

In another alternative embodiment of the present invention, the reactiveprecursor is administered to the afterglow of said plasma together withan aerosol comprising a biomolecule, both of which are deposited andimmobilized onto a sample surface which is positioned in the sameafterglow during the depositing step.

DETAILED DESCRIPTION OF THE INVENTION

The present bio-engineered materials are envisioned to havebio-recognition sites designed to specifically interact with otherbiological or non-biological species of interest. The present inventionallows to design and construct robust bio-engineered surfaces by cold,atmospheric plasma treatment, which allows the binding of all kinds ofbiomolecules to surfaces in a direct way without using chemical linkersthat can change the configuration and activity of biomolecules or thatmay lead to high costs and problems concerning homogeneity. Thistechnology can pave the way to a whole new realm of future applicationsin the medical, chemical, environmental, food, materials and many otherindustrial sectors, including but not limited to:

-   -   Biosensors for large and small-scale applications like for        instance the detection of pollutants (dioxins, pseudo-estrogenic        substances, antibiotics, micro-pollutants, etc. e.g. in water        and air), biomedical diagnostics, toxicity tests etc.;    -   Labs-on-a-chip: the low energy barrier to mobility in the plane        of the surface can be used to facilitate complex reactions that        require a cluster of different proteins, including applications        in the field of molecular biology;    -   Bio-mimetic materials e.g. for implants (mimicking biomolecular        recognition);    -   Solar-cells based on immobilised photosensitive charge transfer        proteins;    -   Non-fouling surfaces for medical diagnostics, heat exchangers,        and food processing equipment;    -   Anti-microbial coatings for (medical) textile, plastics for        medical applications, food packaging;    -   Surfaces for directing controlled drug release;    -   Intelligent materials/textiles, e.g. by incorporating proteins        in conducting plasma polymer coatings, which may allow        transmission of a biological signal to a processor;    -   Templates for extra-corporeal and/or in-vivo growth of        functional tissues;    -   Bio-induced crystalline morphologies: biomolecules ordered on a        surface may induce mineralisation and the morphologies formed        differ from the classical ones. Such mineral surfaces may find        applications in materials development and micro-electronics;    -   Conducting coatings based on conducting proteins (like e.g.        cytochrome C en bovine serum albumin);    -   Bio-catalysis applications e.g. biodegradation of very        recalcitrant molecules in wastewater and removal of        micro-pollutants, catalysis of very specific biochemical        reactions for producing high value chemical compounds (e.g.        chiral compounds).

Stable solutions of biomolecules are administered to a cold atmosphericplasma together with a plasma polymer precursor, either a gas or aliquid. The biomolecules such as proteins, enzymes, nucleic acids andsugars can be in aqueous solution or in a precursor solution. Ifnecessary, aerosols of mixtures or mixtures of different aerosols can beadded to the plasma, possibly together with gaseous precursors.Alternatively, stable solutions of biomolecules are arranged onto thesurface of a sample prior to applying a thin polymer layer on saidsurface by a cold atmospheric plasma treatment with either liquid orgaseous precursor molecules. It is important to incorporate thebiomolecules in a polymer coating in such a way that at least part ofthe biological activity or structure is retained. The present inventionconstitutes a one-step process. Furthermore, any substrate, of any formor material, can be coated with biomolecules using the method of thepresent invention.

A major advantage of the present invention is its ability to treatmaterials in a cost-effective way and at a large scale, which is notfeasible with the current state-of-the-art technology.

The method of immobilisation according to the present inventioncomprises the incorporation of biomolecules, and proteins in particular,in thin plasma polymerised coatings. For this purpose, solutionscontaining these proteins or other biomolecules will be administered toa cold atmospheric plasma together with either liquid or gaseous polymerprecursors. Alternatively the solutions containing these proteins can bearranged onto the surface of a substrate prior to administering thesample to a cold, atmospheric plasma together with either liquid orgaseous polymer precursors. The preferred plasma configuration to beused in practising this invention is the dielectric barrier discharge(DBD), which consists of a uniform glow. Immobilisation of biomoleculesis not feasible with the well-established vacuum or low pressure RF(13.56 MHz) plasma technology for a number of reasons but mainly becauseof the presence of highly energetic species in the plasma which causeconsiderable damage to proteins or may even destroy them. In addition,processing of proteins and protein solutions is impracticable undervacuum conditions.

Plasma processing at atmospheric pressure is a relatively newtechnology—the first reports date from 1990—and it offers manyadvantages over vacuum plasma technology, including the ability to workin-line, the significantly lower process costs and the compatibilitywith virtually any type of substrate material. The most importantfeature of atmospheric pressure plasmas in this context is however theabsence of highly energetic species in the plasma. While complexprecursor molecules get fractured when exposed to vacuum plasma, theyretain their structure to a high extent in atmospheric pressure plasmas.The latter phenomenon is attributed to the reduced mean free path lengthof the active species due to the presence of high amounts of gasmolecules. Accordingly this new technology also allows the incorporationof biomolecules into coatings with only minor modifications. Solutionscontaining biomolecules/proteins, either aqueous or with solvents added,can be administered to the plasma as an aerosol together with a liquidor gaseous hydrocarbon or hybrid organic/inorganic molecule polymerprecursor. Accordingly, biomolecules present in the droplets may beincorporated into thin plasma polymer coatings where they are exposed tothe surface and exhibit their activity. Alternatively, the solutionscontaining biomolecules/proteins are applied onto the surface of asample prior to administering them to a cold, atmospheric plasma, wherea thin layer with a thickness of a few nanometers is deposited on top ofthe biomolecules. The incorporation of biomolecules may be accomplishedphysically (by embedding) or by covalent linking, depending on thereaction conditions and the type of precursor used. During this process,proteins will not be forced to change their conformation in order tobind to a surface because the coating, preferably a coating with a highwater content, will be formed around the proteins, thus stabilising andprotecting them. It remains however important that the orientation ofthe proteins near the surface allows them to expose their biologicallyactive sites or that the cross-link density of the plasma polymer issufficiently low to allow diffusion of the matching substrates tocompletely embedded proteins. Precursors that contain functional groupslike amines and carboxyls will chemically bind to biomolecules whilethis is less likely to occur with precursors like alkanes. In the lattercase embedding of proteins in a coating may occur. The precursorsinclude organic molecules (like acrylic compounds, alkanes, alkenes,etc.) and organic/inorganic hybrid molecules (like HMDSO and TEOS).

Moreover, apart from the presence of low energy radicals, reactionconditions in cold, non-equilibrium plasmas are very mild: lowtemperature (room temperature up to 60° C.) and ambient pressure. Sofar, no literature or patents have been published on the manufacture ofsimilar biofunctional coatings by atmospheric pressure plasmatechnology.

EXAMPLE 1

A plasma discharge at atmospheric pressure is obtained between twohorizontally placed parallel electrodes with a size of 45×45 mm, bothcovered with an alumina (Al₂O₃) plate of 2 mm thickness. The distancebetween the covered electrodes is 2 mm. The top electrode is grounded.The bottom electrode is connected to a variable frequency AC powersource (ENI, model RPG-50). The frequency of the AC power source is setat 2 kHz. In order to perform tests in a controlled environment, theelectrode configuration is mounted in a closed chamber that is evacuatedand subsequently filled with the carrier gas before deposition isstarted.

Helium is used as carrier gas. The flow rate of the carrier gas iscontrolled by a mass flow controller and set at 20 l/min.Hexamethyldisiloxane (HMDSO) is used as reactive precursor. It is addedto the inert carrier gas in the form of an aerosol. Another aerosol,containing an aqueous solution of streptavidin, is added simultaneouslyto the plasma. The deposition time is set at 1 min. Coating depositionis observed at the surface of both electrodes and on the substratesattached to these electrodes. The thickness of the coatings equals 175nm. The presence of streptavidin in the plasma polymer coating obtainedand the ability of streptavidin to bind to fluorescently labelled biotinafter immobilisation were evaluated using fluorescence microscopy. Afterusing fluorescently labelled biotin binding-assay, a signal could beobserved, which indicates that streptavidin was immobilized into thecoating, while retaining at least part of its binding activity.

EXAMPLE 2

A cold, atmospheric pressure plasma discharge is obtained between twohorizontally placed parallel electrodes with a size of 8×15 cm, bothcovered with float glass plate of 3 mm thickness. The distance betweenthe electrodes is 2 mm. The bottom electrode is grounded and connectedto a Peltier element which can provide cooling to room temperature, ifnecessary. The Peltier element is in turn connected to a cooling finwhich is cooled by a fan. The top electrode is connected to a variablefrequency AC power source. An AC-field of 8 kHz and 20 kV is applied tothe electrodes.

Helium is used as a carrier gas. The flow rate of the carrier gas iscontrolled by a mass flow controller and set at 6 l/min. Acetylene isused as reactive precursor. It is mixed with the inert carrier gas andadministered to the plasma at a flow rate of 0.3 l/min. An aerosol,containing an aqueous solution of avidin, is added simultaneously to theplasma. The deposition time is set at 30 seconds. A coating is depositedon the surface of both electrodes and on the glass and siliconsubstrates attached to the electrodes. The thickness of the coatingequals 25 nm as determined by scanning electron microscopy (SEM)analysis of cross-sections of the coated silicon substrates. Thepresence of avidin in the plasma polymer coating obtained and theability of avidin to bind to fluorescently labelled biotin afterimmobilisation were evaluated using fluorescence microscopy. After usingfluorescently labelled biotin binding-assay, a signal could be observed,which indicates that avidin was immobilized into the coating, whileretaining at least part of its binding activity. Grazing-incidencesmall-angle-X-ray scattering analysis (GISAX) was carried out in orderto obtain information on the structure and size of the immobilisedavidin. Apparently at least part of the immobilized avidin has retainedits original structure and shape, and thus its activity.

EXAMPLE 3

The method described in example 2 was repeated using a liquid precursor,being pyrrole, instead of acetylene. Pyrrole was administered to theplasma zone as an aerosol. Again, coating deposition was observed on thesurface of both electrodes and on the glass and silicon substratesattached to their surface. The coating thickness equaled 35 nm after 30seconds of deposition.

EXAMPLE 4

The reactor set-up described in example 2 was used for theimmobilization of bovin serum albumin (BSA). Helium was administered tothe plasma zone at a flow rate of 6 l/min. Pyrrole is used as reactiveprecursor. It is added to the inert carrier gas as an aerosol. Anotheraerosol, containing an aqueous solution of BSA is simultaneously addedto the plasma. An AC-field of 2 kHz and 20 kV is applied to theelectrodes. The deposition time is set at 30 seconds. A coating isdeposited on the surface of both electrodes and on the glass and siliconsubstrates attached to the electrodes. The thickness of the coatingequals 35 nm as determined by scanning electron microscopy (SEM)analysis of cross-sections of the coated silicon substrates.Grazing-incidence small-angle-X-ray scattering analysis (GISAX) wascarried out in order to obtain information on the structure and size ofthe immobilized BSA. Apparently, a substantial part of the immobilizedBSA has retained its original structure and shape, and thus itsactivity.

EXAMPLE 5

A solution of bovin serum albumin (BSA) is spread out onto a glasssubstrate. After drying the sample for 12 hours at room temperature, itis placed on the lower electrode of the set-up described in example 2.Helium and acetylene are administered to the zone between the electrodesat a flow rate of 6 and 0.3 l/min, respectively. After 10 seconds ofdeposition, a layer with a thickness of 3 to 5 nm was obtained. Thesample was analyzed by means of grazing-incidence small-angle-X-rayscattering analysis (GISAX) and apparently, BSA has retained itsoriginal structure and size to a high extent after this type oftreatment.

1. A method for immobilising a biomolecule on a sample surface bygenerating and maintaining a cold atmospheric pressure plasma, saidmethod comprising the steps of: introducing a sample in the spacebetween a first and a second electrode, a mixed atmosphere being presentbetween said electrodes, applying an alternating voltage to said firstand second electrode for generating and maintaining a plasma in thevolumetric space between said electrodes, said voltage alternatingbetween a positive voltage for said first electrode and a zero voltagefor said second electrode, and a zero voltage for said first electrodeand a negative voltage for said second electrode, and depositing acoating on a surface of said sample, wherein said mixed atmospherecomprises a reactive precursor and an aerosol comprising thebiomolecule, and wherein said reactive precursor is deposited and saidbiomolecule is immobilised during the depositing step.
 2. The methodaccording to claim 1 wherein the reactive precursor is a gas or a liquidin the form of an aerosol.
 3. A method for immobilizing a biomolecule ona sample surface by generating and maintaining a cold atmosphericpressure plasma, said method comprising the steps of: introducing asample in the space between a first and a second electrode, a mixedatmosphere being present between said electrodes, applying analternating voltage to said first and second electrodes for generatingand maintaining a plasma in the volumetric space between saidelectrodes, said voltage alternating between a positive voltage for saidfirst electrode and a zero voltage for said second electrode, and a zerovoltage for said first electrode and a negative voltage for said secondelectrode, and depositing a coating on a surface of said sample, whereina reactive precursor is deposited and a biomolecule is immobilisedduring the depositing step, and wherein the reactive precursor isadministered to the afterglow of said plasma together with an aerosolcomprising a biomolecule, both of which are deposited and immobilizedonto a sample surface which is positioned in the same afterglow duringthe depositing step.
 4. The method according to claim 1, wherein thebiomolecule is selected from the group consisting of a protein, apolynucleotide, a sugar, a lipid, a growth factor, a hormone.
 5. Themethod according to claim 1, wherein the reactive precursor is selectedfrom the group consisting of a hydrocarbon, a fluorinated hydrocarbonand an organometallic compound or a combination thereof.
 6. The methodaccording to claim 1, wherein the mixed atmosphere comprises helium,argon, nitrogen, air, carbon dioxide, ammonium or a combination thereof.7. The method according to claim 1, wherein the sample comprises metal,ceramic or plastic materials, woven or non-woven fibres, natural fibresor synthetic fibres or powders.
 8. A method according to claim 1,wherein the electrodes are cooled to temperatures between 0° C. and 100°C. 9-10. (canceled)